Hirudin insensitive thrombin-stimulated platelet release

It has been previously proposed that platelet stimulation may involve two platelet-thrombin complexes: an initial platelet-thrombin complex (P-T) which is converted to an activated platelet-thrombin complex (P*-T). By using the release of radioactive serotonin as a measure of thrombin stimulation, we have demonstrated that under appropriate conditions, a hirudin sensitive and a hirudin insensitive complex can be differentiated. At short platelet-thrombin preincubation times (0-2 minutes) at 4 degrees C, in a buffer containing 18.7 mM phosphate, added hirudin almost completely inhibited the release of radioactive serotonin obtained upon subsequent warming to 37 degrees C (only the hirudin sensitive complex exists). If platelets were preincubated with thrombin for longer periods of time (30 minutes), hirudin became less effective in inhibiting the release obtained upon subsequent warming to 37 degrees C. The same results were obtained whether or not the platelets were washed after incubation at 4 degrees C and before warming to 37 degrees C. We postulate that this change in hirudin sensitivity may reflect a slow conversion of the first platelet-thrombin complex (P-T) to an activated platelet-thrombin complex (P*-T) which can undergo release upon warming. This transition appears to be much faster in acetatetris buffer since at short platelet-thrombin incubation times at 4 degrees C, added hirudin had little or no effect on the release obtained upon warming to 37 degrees C. The difference in the ability of hirudin to inhibit thrombin-induced release in the two buffers was shown to depend upon the presence of phosphate and on variations in ionic strength, and not due to a change in the inhibition constant (Ki) for hirudin.